CD6 is an important cell surface protein predominantly expressed by human T cells and a subset of B cells, as well as by some B cell chronic lymphocytic leukemias and neurons (see, e.g., Aruffo et al., J. Exp. Med., 174:949 (1991); Kamoun et al., J. Immunol. 127:987 (1981); Mayer et al., J. Neuroimmunol. 29:193 (1990)). CD6 is a member of a large family of proteins characterized by having at least one domain homologous to the scavenger receptor cysteine-rich domain (SRCR) of type I macrophages (Matsumoto, et al., J. Exp. Med., 173:55 (1991) and Resnick et al., Trends Biochem. Sci., 19:5 (1994)). Other members of this family include CD5 (Jones et al., Nature, 323:346 (1986); cyclophilin C (Friedman et al., PNAS 90:6815 (1993)); complement factor I, which binds activated complement proteins C3b and C4b (Goldberger, et al., J. Biol. Chem., 262:10065 (1987)); bovine WC-1 expressed by xcfx84/xcex4 T cells (Wijingaard et al., J. Immunol., 149:3273 (1992)); and M130 (Law et al., Eur J. Immunol., 23:2320 (1993)), a macrophage activation marker.
Blocking studies using anti-CD6 monoclonal antibodies (mAbs) suggest that CD6 plays an important role in T cell development by regulating T cell adhesive interactions with thymic epithelial (TE) cells (Patel et al., J. Exp. Med. 181:1563-1568 (1995)). Additional studies have shown that CD6 can function as an important accessory molecule in T cell activation. For example, certain anti-CD6 mAb are directly mitogenic for T cells (Gangemi et al., J. Immunol., 143:2439 (1989) and Bott et al., Int. Immunol. 7:783 (1993), whereas others are able to co-stimulate T cell proliferation in conjunction with anti-CD3, anti-CD2 or PMA (Gangemi et al., J. Immunol., 143:2439 (1989); (Morimoto et al., J. Immunol., 140:2165-2170 (1988); and (Osorio et al., Cell. Immunol., 154:23 (1994)). Yet additional evidence of the role of CD6 in T cell activation comes from studies showing that CD6 becomes hyperphosphorylated on Ser and Thr residues (Swack et al., Mol. Immunol. 26:1037-1049 (1989); Swack et al., J. Biol. Chem.266:7137 (1991); Cardenas et al., J. Immunol., 145:1450-1455 (1990)) and phosphorylated on Tyr residues (Wee et al., J. Exp. Med., 177:219-223 (1993)) following T cell activation. These and other studies implicate CD6 as an important modulator of both immature and mature T cell function in vivo, affecting both T cell activation and signal transduction.
The extracellular domain of the mature CD6 protein is composed of three SRCR domains (hereinafter designated CD6D1, CD6D2, and CD6D3, with CD6D3 corresponding to the membrane proximal SRCR domain) followed by a short 33-amino-acid stalk region. These extracellular domains are anchored to the cell membrane via a short transmembrane domain followed by a cytoplasmic domain of variable length (Aruffo et al., J. Exp. Med., 174:949 (1991)).
Studies using CD6-immunoglobulin fusion proteins, containing selected extracellular domains of CD6 fused to human IgG1 constant domains (CD6-Rgs), led to the identification and cloning of a CD6 ligand, designated xe2x80x9cactivated leukocyte cell adhesion moleculexe2x80x9d (ALCAM) (Wee, et al., Cell. Immunol., 158:353-364, (1994); Patel, et al., J. Exp. Med. 181:1563-1568 (1995); Bowen et al., J. Exp. Med., 181:2213-2220 (1995). ALCAM is a member of the immunoglobulin supergene family and may be a human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP (Pourquie et al., PNAS, 89:5261-5265 (1992); Tanaka et al., Neuron, 535-545 (1991); and Burns et al., Neuron, 209-220 (1991)) and the rat protein KG-CAM (Peduzzi et al., Brain Res., 640:296-307 (1994)). In the chicken, BEN/SC-1/DM-GRASP is able to mediate homophilic interactions, and has been shown to be involved in neurite outgrowth in the nervous system.
In addition to being expressed by neurons, ALCAM is expressed by human TE cells and a variety of other cell types (Patel et al., J. Exp. Med., 181:1563 (1995)) and transiently expressed by activated leukocytes (Bowen et al., J. Exp. Med., 181:2213 (1995)). Notably, cell adhesion assays demonstrated that CD6-ALCAM interactions are in part responsible for mediating thymocyte binding to TE cells (Bowen et al., J. Exp. Med., 181:2213-2220 (1995)). Analysis of the in vitro kinetics of human ALCAM expression showed that its expression by mitogen activated peripheral blood T cells peaks 72 hours after stimulation and returns to undetectable levels between 5 and 8 hours. BEN/SC-1/DM-GRASP of the chicken is also expressed by activated T cells (Corbel et al., Cell Immunol. 141:99 (1992)) and hemopoietic progenitor cells and has been shown to mediate heterophilic interactions with NgCAM and other proteins (DeBernardo et al., J. Cell. Biol., 133:657 (1996)). (Corbel et al., PNAS, 93:2844 (1996)). Studies of the role of CD6/ALCAM interactions in T cell regulation have shown that this receptor-ligand pair is able to mediate the adhesion of CD6 expressing cells to thymic epithelial cells (Bowen et al., J. Exp. Med., 181:2213 (1995)). This and other evidence suggests that CD6/ALCAM interactions are important for modulating T cell development and activation.
Although the above findings indicate that CD6/ALCAM interactions play an important role in regulating T cell development and activation, there remains a clear need in the art for further discovery and characterization of CD6, and human CD6 in particular, especially with regard to its interactions with ALCAM. More specifically, there is a need in the art for further characterization of hCD6 structural elements that mediate hCD6/ALCAM binding interactions, and for specific tools, such as hCD6 binding agents, that can modulate hCD6/ALCAM interactions. Such tools would be useful in various diagnostic uses, ex vivo treatments, and in vivo therapeutic methods, for example for diagnosing CD6-mediated responses linked to disease states in patients, for conducting ex vivo affinity removal of CD6+ cells from transplant materials, and for providing in vivo modulating agents, e.g., inhibitors or enhancers, of CD6-mediated T cell activation, to modulate inflammatory and autoimmune responses in patients. The present invention addresses these needs and provides additional advantages that will become apparent from the description which follows.
The invention provides antibodies and other binding agents that bind specifically to SRCR domains of human CD6 (hCD6). In preferred aspects of the invention, antibodies and other immunoglobulins, including native and artificially modified antibodies and antibody fragments, are provided that bind specifically to human CD6 SRCR domain 3 (CD6D3) or human CD6 stalk domain (CD6S) and inhibit activated leukocyte cell adhesion molecule (ALCAM) binding to CD6.
In more detailed aspects of the invention, anti-human CD6 binding agents are selected from exemplary, native monoclonal antibodies identified hereinbelow and included within one of eight CD6 binding subgroups designated as Group 1 (exemplified by mAb 5D4); Group 2 (exemplified by mAb 10A5); Group 3 (exemplified by mAb 16A3); Group 4 (exemplified by mAb 7H6); Group 5 (exemplified by mAb 15B12); Group 6 (exemplified by mAbs 7C7 and 13C3); Group 7 (exemplified by mAbs 5E8 and 8A7); or Group 8 (exemplified by mAbs 10D1 and 12A5). Alternatively, anti-human CD6 binding agents may be selected from modified immunoglobulins, for example humanized antibodies, site directed mutagenized antibodies, or chemically or recombinantly produced antibody fragments, that exhibit substantial amino acid sequence identity to corresponding native antibodies and retain substantially the same CD6 binding specificity as the corresponding native antibody.
In other embodiments of the invention, screening methods are provided for identifying additional binding agents that specifically bind hCD6. These methods entail contacting a reference anti-hCD6 monoclonal antibody that binds specifically to human CD6 SRCR domain 3 (CD6D3) or human CD6 stalk domain (CD6S) and inhibits ALCAM binding to hCD6 with a target species comprising one or more hCD6 domains selected from CD6D2, CD6D3 and CD6S in the presence of a putative competitor test binding agent. This step of contacting is conducted under conditions suitable for complex formation between the reference antibody and the target species in the absence of the test binding agent. Next, complex formation between the reference antibody and the target species in the presence of the test binding agent is detected as an indicator of specific binding activity of the test binding agent to CD6D3 or CD6S. This screening method is useful for high throughput screening of, e.g., peptide and small molecule libraries to identify and characterize additional hCD6 binding agents. Preferred antibodies for these assays are also selected from the CD6 binding subgroups Group 1 (5D4); Group 2 (10A5); Group 3 (16A3); Group 4 (7H6); Group 5 (15B12); Group 6 (7C7, 13C3); Group 7 (5E8, 8A7); or Group 8 (10D1, 12A5), or from fragments or other artificially modified forms of these antibodies.
In related aspects of the invention, the foregoing screening methods are adapted by the additional steps of contacting ALCAM with the target species in the presence of the test binding agent under conditions suitable for ALCAM binding to the target species. Subsequently, complex formation is detected between ALCAM and the target species as an indicator of activity of the test binding agent for modulating ALCAM/CD6 binding. Preferred test binding agents for selection by such screening methods include peptide mimetics of a complementarity determining region (CDR) of the reference antibody, as well as other peptides and small molecular species that may be selected for their ability to modulate CD6/ALCAM binding interactions.
In other aspects of the invention, methods are provided for modulating inflammatory or autoimmune responses in patients, for example methods for inhibiting adverse responses associated with multiple sclerosis or transplant rejection. These methods include administration to a patient of a therapeutically or pharmaceutically effective amount of an anti-CD6 binding agent that binds specifically to human CD6 SRCR domain 3 (CD6D3) or human CD6 stalk domain (CD6S) and inhibits ALCAM binding to hCD6. Preferred anti-CD6 binding agents for use in these methods are monoclonal antibodies, including humanized monoclonal antibodies, as well as modified immunoglobulins such as antibody fragments and mutagenized forms of native antibodies having substantial amino acid sequence identity with a corresponding native antibody, and sharing substantially the same binding specificity therewith.
In yet additional aspects of the invention, diagnostic compositions and methods are provided for detecting CD6, CD6+ cells, and/or CD6-mediated activity, for example CD6 activity related to T cell activation, in in vitro and in vivo assays. These methods likewise employ anti-CD6 binding agents that bind specifically to human CD6 SRCR domain 3 (CD6D3) or human CD6 stalk domain (CD6S) and/or inhibit ALCAM binding to hCD6.